Overexpression of c-kit rescues postnatal neurogenesis defect and adult depressive-like phenotype in <i>camk2a-Hap1</i> KO mice.

<p>(<b>A</b>) Neuro-2A cells were infected with AAV-c-kit or control virus for 72 hours, and the expression of c-kit was analyzed by western blot. The top band may be the precursor of c-kit, which is not present in the brain lysate. (<b>B</b>) AAV-c-kit was stereotaxically injected into P3 mouse hippocampus. Two weeks later, the expression of c-kit in the hippocampus was examined by immunostaining, which showed robust expression of c-kit in the hilus and subgranular zone of the DG. AAV-GFP injection served as a control. (<b>C-D</b>) C<i>amk2a-Hap1</i> KO and control mice at P3 were injected with AAV-c-kit or control GFP virus into the hippocampus. BrdU was i.p. injected into these mice at P17, and 24 hours later, BrdU staining (C) and stereological quantification of BrdU+ cells (D, n = 4–6 per group) were performed, which revealed enhanced hippocampal neurogenesis by the overexpression of c-kit. (<b>E-F</b>) 2–3 months old <i>Camk2a-Hap1</i> KO and control mice, which were injected with AAV-c-kit or control virus into the hippocampus at P3, were subjected to the FST (E) and TST (F). Both tests demonstrated a rescue effect of c-kit on adult depressive-like behavior in <i>camk2a-Hap1</i> KO mice. n = 10–20 per group. All error bars represent SEM. n.s., not significant, *p<0.05, **p<0.01, ***p<0.001.</p

Postnatal Loss of Hap1 Reduces Hippocampal Neurogenesis and Causes Adult Depressive-Like Behavior in Mice

<div><p>Depression is a serious mental disorder that affects a person’s mood, thoughts, behavior, physical health, and life in general. Despite our continuous efforts to understand the disease, the etiology of depressive behavior remains perplexing. Recently, aberrant early life or postnatal neurogenesis has been linked to adult depressive behavior; however, genetic evidence for this is still lacking. Here we genetically depleted the expression of huntingtin-associated protein 1 (Hap1) in mice at various ages or in selective brain regions. Depletion of Hap1 in the early postnatal period, but not later life, led to a depressive-like phenotype when the mice reached adulthood. Deletion of <i>Hap1</i> in adult mice rendered the mice more susceptible to stress-induced depressive-like behavior. Furthermore, early Hap1 depletion impaired postnatal neurogenesis in the dentate gyrus (DG) of the hippocampus and reduced the level of c-kit, a protein expressed in neuroproliferative zones of the rodent brain and that is stabilized by Hap1. Importantly, stereotaxically injected adeno-associated virus (AAV) that directs the expression of c-kit in the hippocampus promoted postnatal hippocampal neurogenesis and ameliorated the depressive-like phenotype in conditional <i>Hap1</i> KO mice, indicating a link between postnatal-born hippocampal neurons and adult depression. Our results demonstrate critical roles for Hap1 and c-kit in postnatal neurogenesis and adult depressive behavior, and also suggest that genetic variations affecting postnatal neurogenesis may lead to adult depression.</p></div

Overexpression of c-kit rescues postnatal neurogenesis defect and adult depressive-like phenotype in <i>camk2a-Hap1</i> KO mice.

<p>(<b>A</b>) Neuro-2A cells were infected with AAV-c-kit or control virus for 72 hours, and the expression of c-kit was analyzed by western blot. The top band may be the precursor of c-kit, which is not present in the brain lysate. (<b>B</b>) AAV-c-kit was stereotaxically injected into P3 mouse hippocampus. Two weeks later, the expression of c-kit in the hippocampus was examined by immunostaining, which showed robust expression of c-kit in the hilus and subgranular zone of the DG. AAV-GFP injection served as a control. (<b>C-D</b>) C<i>amk2a-Hap1</i> KO and control mice at P3 were injected with AAV-c-kit or control GFP virus into the hippocampus. BrdU was i.p. injected into these mice at P17, and 24 hours later, BrdU staining (C) and stereological quantification of BrdU+ cells (D, n = 4–6 per group) were performed, which revealed enhanced hippocampal neurogenesis by the overexpression of c-kit. (<b>E-F</b>) 2–3 months old <i>Camk2a-Hap1</i> KO and control mice, which were injected with AAV-c-kit or control virus into the hippocampus at P3, were subjected to the FST (E) and TST (F). Both tests demonstrated a rescue effect of c-kit on adult depressive-like behavior in <i>camk2a-Hap1</i> KO mice. n = 10–20 per group. All error bars represent SEM. n.s., not significant, *p<0.05, **p<0.01, ***p<0.001.</p

<i>camk2a-Hap1</i> conditional KO mice also display adult depressive-like behavior.

<p>(<b>A</b>) A schematic of conditional <i>Hap1</i> KO in <i>camk2a</i>-Cre-expressing neurons in mice. (<b>B</b>) Western blotting result showing that the Hap1 protein level is decreased in the forebrain of <i>camk2a-Hap1</i> KO mice at P7. Note that the decrease in the hippocampus is much more dramatic than in the hypothalamus. Hip: hippocampus; Ctx: cortex; Str: striatum; Hyp: hypothalamus. (<b>C</b>) Body weight recording of <i>camk2a-Hap1</i> KO and control mice. Daily body weight recording was done for the first month using 2 litters of mice. At 4 months of age, 7 litters were used. (<b>D</b>) FST and TST revealed depressive-like behavior in 2-month old <i>camk2a-Hap1</i> KO mice. (<b>E</b>) Sucrose preference was assessed during acclimation and in acute and overnight tests. Two-month-old <i>camk2a-Hap1</i> KO mice showed reduced preference compared to controls. Fluid intake was also measured for the overnight test. <i>Camk2a-Hap1</i> KO mice consumed significantly less sucrose solution than controls. All error bars represent SEM. n.s., not significant, *p<0.05, **p<0.01.</p

Early postnatal Hap1 depletion leads to reduced hippocampal neurogenesis.

<p>(<b>A</b>) <i>Hap1</i> P1 KO and control mice were injected with BrdU at P6, and then perfused at P7 for immunostaining of BrdU in the DG of the hippocampus. P1 KO mice showed significantly fewer BrdU+ cells in the DG. Scale bar: 40 μm. (<b>B</b>) Stereological quantification of BrdU+ cells in (A). n = 4 per genotype. (<b>C, D</b>) Stereological quantification of BrdU+ cells in the DG of <i>Hap1</i> P1 KO (C) or <i>Hap1</i> P21 KO (D) and control mice at P34 that were injected with BrdU at P33. n = 4 for control, n = 5 for KO. (<b>E</b>) Stereological quantification of BrdU+ cells in the DG of P34 <i>Camk2a-Hap1</i> KO and control mice that were injected with BrdU at P33. n = 5 for control, n = 4 for KO. (<b>F</b>) Quantification of the ratios of NeuN+ (left panel) and GFAP+ cells (right panel) among BrdU+ cells. n = 4 per genotype. Double staining images of NeuN+/BrdU+ and GFAP+/BrdU+ cells are also presented. Arrows indicate double-positive cells. Scale bar: 10 μm. All error bars represent SEM. *p<0.05, **p<0.01, ***p<0.001.</p

Adult Hap1 expression reduces the stress-induced defective neurogenesis in the hippocampus and depressive-like behavior.

<p>(<b>A</b>) One-year-old <i>Hap1</i> P21 KO and control mice were subjected to 4 h restraint stress for 7 consecutive days. BrdU was i.p. injected in the final 3 days of the treatment. Immunostaining of BrdU in the DG was performed 1 day after the final treatment. Scale bar: 50 μm. The stressed groups showed fewer BrdU+ cells than non-stressed groups, and the decrease was more dramatic for stressed KO mice than stressed controls. (<b>B</b>) Stereological quantification of BrdU+ cells in (A). n = 3–4 mice per group. (<b>C</b>) The forced swim test was conducted 1 day after the final treatment. n = 9–11 per group. While stressed control mice did not exhibit depressive-like behavior compared to non-stressed controls, loss of Hap1 caused stressed KO mice to display significant depressive-like behavior compared with stressed control and non-stressed KO mice. All error bars represent SEM. *p<0.05, **p<0.01.</p

Oral administration of BBR reduced mutant Htt aggregation and increased autophagy in transgenic N171-82Q mice.

<p><b>(A)</b> Fluorescent immunohistostaining of mouse brain slices from transgenic N171-82Q HD mice and WT siblings, orally gavaged with 40 mg/kg BBR or vehicle daily. <b>(B)</b> Western blotting of the cortex samples of 3 mice per group (HD+BBR vs. HD+vehicle) showing mutant Htt aggregates in the stacking gel and P62. Non-specific immunoreactive bands are also shown on the blots. Actin was used to show the internal control protein on western blot. <b>(C)</b> Densitometry analysis of above Western blots. The data are mean±SE (n = 3). <b>(D)</b> Quantitative RT-PCR analysis of transgenic mutant Htt mRNA in the cortex of the BBR-treated or untreated N171-82Q mice. n = 3 mice per group. Data are presented as mean CT±SEM. <b>(E)</b> Western blotting of WT mouse cortex samples (3-mouse pool per group) after the mice had been orally gavaged with 40 mg/kg BBR or vehicle daily for 24 weeks and sacrificed 4 h after the last BBR administration. <b>(F)</b> Densitometry analysis of Western blots in (E) showing the ratio of P62 to actin. The quantitative data are presented as mean±SE.</p

Early postnatal depletion of Hap1 leads to depressive-like behavior in adult mice.

<p>(<b>A</b>) A schematic of the TM-inducible Cre/loxP system to delete <i>Hap1</i> in mice. (<b>B-F</b>) Forced swim and tail suspension tests (FST and TST) were performed at 3 months of age for <i>Hap1</i> P1 (B, P1 KO), P10 (C, P10 KO), P15 (D, P15 KO) and P21 (E, P21 KO) KO mice, and 4 months of age for mice that had <i>Hap1</i> gene deleted at 2 months old (F, adult KO). Note that early postnatal Hap1 depletion, but not late postnatal or adult depletion, led to increased immobility in the FST and TST. (<b>G</b>) Locomotor activities of 2-month-old <i>Hap1</i> P1 KO and control mice were monitored and recorded for 24 h. The KO mice showed a general reduction in locomotor activity. All error bars represent SEM. *p<0.05, **p<0.01. In (G), two-way (genotype and time) ANOVA and post hoc tests were performed to determine statistical significance.</p

Oral administration of BBR ameliorates neurological symptoms in transgenic N171-82Q mice.

<p><b>(A, B)</b> Rotarod test of motor coordination of N171-82Q transgenic mice and WT siblings, orally gavaged with 40 mg/kg BBR or vehicle daily. <b>(C, D)</b> Grip strength test on transgenic N171-82Q mice and WT siblings treated with BBR or vehicle. <b>(E)</b> Balance beam test on 18-week-old N171-82Q and WT mice that had been treated with BBR or vehicle. <b>(F)</b> Survival curve for N171-82Q mice and WT siblings treated with BBR or vehicle. (<b>G</b>) The BBR-treated N171-82Q group survived 15 days longer than their untreated HD siblings. <b>(H)</b> Body weight of N171-82Q mice and WT siblings treated with or without BBR. The quantitative data are presented as mean±SE. *p<0.05. **p<0.01. ***p<0.001, n = 7 mice per group.</p

BBR increases autophagic activity in cultured cells.

<p><b>(A)</b> Western blotting of LC3B in HEK293 cells transfected with or without Htt and treated with BBR (0, 5, 25, 50, or 100 μM). <b>(B)</b> Densitometry analysis of the ratios of LC3-I to LC3-II in above Western blots. <b>(C)</b> Western blotting of Htt-transfected HEK293 cells treated with or without 50 μM BBR or bafilomycin (BFA), an autophagy inhibitor. Antibody to P62, which decreases when autophagy activates, was used to confirm the altered activity of autophagy. <b>(D)</b> Densitometry analysis of the ratios of aggregated Htt or P62 to actin in Western blots in (C). The quantitative data are presented as mean±SE.</p